Nucleic acids encompass both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA, present in all nucleated cells, carries the information needed to direct the synthesis of every protein in the body. A single alteration in the correct sequence of the four DNA bases (adenine, thymidine, guanine, and cytosine) may result in a defective protein. Depending upon the protein and the affected organism, the defect may range from inconsequential to life-threatening, or may be of intermediate severity. Diseases as diverse as cystic fibrosis, some types of cancer, sickle cell anemia, and atherosclerosis are known to result from specific genetic alterations.
RNA, the intermediary between DNA and protein, is the product of transcription of a DNA template. RNA assays are being performed with increasing frequency in research and clinical laboratories. This is due at least in part to the prevalence of RNA viruses such as the human immunodeficiency virus (HIV) that causes AIDS and the hepatitis C virus (HCV), and the development of drugs used in treating infections with RNA viruses. Precise testing for the presence of specific nucleic acid sequences for identification or monitoring of disease is important, and constructs comprising nucleic acids with known sequences are necessary for validation, calibration, and standardization of those tests.
Nucleic acid assays are routinely performed, either manually or by automated instrumentation, in numerous reference and clinical laboratories. A nucleic acid assay may be performed to detect the presence of foreign DNA or RNA, which may indicate infection with a foreign organism. For example, a variety of molecular assays are used to establish the presence and identity of nucleic acids from the human immunodeficiency virus-1 (HIV-1), Chlamydia, and other organisms causing sexually transmitted diseases. An individual""s DNA may also be analyzed to detect, treat, and in some cases prevent genetic disease. Genotype determination of genes for Factor-V Leiden, hereditary hemochromatosis, lipoprotein lipase mutations, and cystic fibrosis have important implications for health management. The Human Genome Project holds the promise of many more examples of medically efficacious genetic diagnostic determinations. The recent discovery of the breast cancer associated gene (BRCA-1) has highlighted both the importance of screening individuals for predisposition to a disease, and also the attendant need for accurate, precise, reproducible, and controlled nucleic acid assays.
Nucleic acid testing of a patient derived specimen is a multi-step process. Failure of any step in the process leads to inaccurate clinical information with potentially serious outcome for the patient. The clinical nucleic acid testing protocol includes amplification of one or more DNA segments, and detection of product by any of a number of techniques including binding and detection of labeled probe, and/or restriction enzyme digestion and/or electrophoresis. The test may fail to give the correct result due to interfering substances, unsuitable reaction conditions, reagent problems or detection system failure. The test may be functioning in most aspects but have lost its sensitivity to detect specific mutations or to detect low levels of a given nucleic acid sequence. Some tests experience interference from unexpected polymorphisms or rare mutations and subsequently yield erroneous results. All of these errors may be detected by testing suitable known reference materials in parallel with the patient specimen. Detection of the expected signal from appropriate quality controls validates the testing process.
Current mutation detection technologies fall into two broad categories. The first group includes mutation-scanning technologies; such as single-strand conformational polymorphism (SSCP), modified double gradient gel electrophoresis (DG-DGGE), heteroduplex analysis (HET), various cleavage assays, and direct sequencing. These procedures are generally too difficult and time-consuming for most diagnostic laboratories. In the second group are methodologies more amenable to high throughput diagnostic testing. These include multiplex allele-specific diagnostic assay (MASDA), amplification refractory mutation system (ARMS), PCR followed by an oligonucleotide ligation assay and sequence-coded separation (PCR/OLA/SCS), PCR-mediated site-directed mutagenesis (PSM), and various versions of forward and reverse allele-specific oligonucleotide (ASO) dot blots. All of these assays begin with amplification of 200 to 500 base pair fragments from genomic DNA. Appropriate reference material for these assays should contain one or more of these genomic nucleic acid fragments.
In the field of molecular pathology and genetic testing, a quality control sample includes a reference DNA or reference RNA of known quantity and quality to evaluate the reliability of all steps of a test. Such reference nucleic acid is ideally as similar as possible to the test sample, is available containing combinations of all relevant mutations and polymorphisms, and also has broad applicability to all test formats. Additionally, the reference nucleic acid should be easily produced, quantitated, and packaged with minimal technical capability. Materials meeting these requirements, however, are not available. Reference materials in use include cultured cell lines and patient-based controls materials such as previously tested DNA. They also include DNA extracted and purified from cell lines or patient based specimens. These materials suffer, however, in that they are expensive, difficult to maintain, and limited with respect to the number of genetic diseases, organisms, and combinations of mutations and polymorphisms that they represent.
The need to rely on patient-derived control material also makes it difficult to provide sufficient reference products to cover the large variety of genetic disorders. This is especially problematic when testing for diseases caused by multiple mutations. For example, cystic fibrosis (CF) is a common hereditary disease affecting 1 in 3200 Caucasian newborns in the United States, but the wide variety of mutant alleles makes it difficult to assemble a comprehensive CF proficiency panel. At the NIST Nucleic Acid Workshop, Wayne Grody, Division of Medical Genetics at UCLA, acknowledged the lack of DNA standards and noted that the CAP/ACMG Biochemical and Molecular Genetics Resource Committee would like to dramatically increase the challenges offered.
Further, the unavailability of widely applicable controls is due in part to the variety of different technologies and techniques currently employed for a given diagnostic determination. For example, genetic determinations currently include the use of the polymerase chain reaction (PCR), the ligase chain reaction (LCR), branched DNA, allele specific hybridization, and direct sequence determination. In addition, so-called xe2x80x9chome brewxe2x80x9d produced primer oligonucleotides, and isotopically labeled or non-radioisotopic based probes are used in a variety of configurations in genetic testing, but without any systematic quality control materials, and hence without any validation.
The aforementioned factors, coupled with the lability of nucleic acids, make it virtually impossible to obtain standard reagents to qualitatively and/or quantitatively assess the overall accuracy, reliability, and efficiency of a laboratory assay.
Cystic fibrosis (CF) is an important genetic disease related to mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Many of the most common disease causing mutations are in exon 10 and exon 11 of the CFTR gene, and thus, genetic screening for these mutations is advantageous for early diagnosis of CF. Genetic testing for CF, as well as many other diseases, typically begins with amplification of the nucleic acid segment of interest (e.g., exon 10 and 11), and therefore, controls for these tests must include the nucleic acid region to be amplified. Quality controls for genetic tests are required by federal law and good laboratory practice, but are currently unavailable commercially for CF testing. The invention described herein fulfills the urgent need for validation materials in a variety of molecular assays, including genetic testing for cystic fibrosis.
The invention includes an isolated control DNA construct comprising a vector portion for expression in a cell and a target nucleic acid comprising two or more nucleic acid fragments wherein each fragment specifies a component associated with at least one of a disease state, an environmental condition, or a biological organism, wherein the component is different from a component specified by any other fragment present elsewhere in the construct, and wherein the 5xe2x80x2-most fragment is linked to the vector portion via a restriction site not present elsewhere in the construct, and wherein the 3xe2x80x2-most fragment is linked to the vector portion via a restriction site not present elsewhere in the construct, and further wherein each the fragment is flanked by a restriction site not present elsewhere in the construct.
In one aspect, each of the fragments are selected from the group consisting of fragments of the same gene, and fragments of different genes.
In another aspect, each of the fragments comprise at least one exon of a gene.
In yet another aspect, the exon further comprises an intronic border fragment.
In a further aspect, each of the fragments are selected from the group consisting of fragments from the same organism and fragments from different organisms.
In yet a further aspect, the exon is a cystic fibrosis transmembrane conductance regulator (CFTR) exon.
In another aspect, the CFTR exon is selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6a, exon 6b, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14a, exon 14b, exon 15, exon 16, exon 17a, exon 17b, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, and exon 24.
In another aspect, the CFTR exon is selected from the group consisting of exon 10 and exon 11.
In yet another aspect, the restriction site is selected from the group consisting of a BssH II site, a Csp45 I site, a Age I site, and a Nco I site.
In a further aspect, the restriction site linking the 5xe2x80x2-most fragment to the vector portion is BssH II.
In yet a further aspect, the 5xe2x80x2-most fragment is CFTR exon 10.
In another aspect, the restriction site linking the 3xe2x80x2-most fragment to the vector portion is Age I.
In a further aspect, the 3xe2x80x2-most fragment is CFTR exon 11.
In another aspect, the restriction site linking the 3xe2x80x2 and 5xe2x80x2 ends of each of the fragments within the construct is Csp45 I.
In yet another aspect, the exon 10 comprises a mutation or polymorphism associated with cystic fibrosis.
In a further aspect, the mutation is selected from the group consisting of a G480C mutation, a DI507 mutation, and a DF508 mutation.
In another aspect, the polymorphism is selected from the group consisting of a F508C polymorphism, a I507V polymorphism, and a I506V polymorphism.
In yet another aspect, the exon 11 comprises a mutation or polymorphism associated with cystic fibrosis.
In further aspect, the mutation is selected from the group consisting of a G542X mutation, a G551D mutation, an R553X mutation, an A559T mutation, and an R560T mutation.
In yet a further aspect, the polymorphism is selected from the group consisting of a F508C polymorphism, a I507V polymorphism, and a I506V polymorphism.
In another aspect, the fragments comprise a nucleic acid selected from the group consisting of a Giardia lamblia nucleic acid, a Cryptosporidium parvum nucleic acid, a human immunodeficiency virus nucleic acid, a hepatitis C virus nucleic acid, a factor V nucleic acid, a Chlamydia trachomatis nucleic acid, a Mycobacterium tuberculosis nucleic acid, a nucleic acid associated with hereditary hemochromatosis, a parvovirus B19 nucleic acid, a lipoprotein lipase gene, a methyltetrahydrofolate reductase gene, a beta cystathionase synthetase nucleic acid, a Factor II nucleic acid, a Factor VII nucleic acid, a Factor VIII nucleic acid, Factor IX nucleic acid, a prothrombin nucleic acid, and a nucleic acid comprising a translocation associated with hematologic disease.
In yet another aspect, the nucleic acid comprising a translocation associated with hematologic disease is a BCR/abl nucleic acid.
The invention includes a method of producing an isolated control DNA construct. The method comprises linking the 5xe2x80x2-most end of a nucleic acid fragment with a 3xe2x80x2end of a vector, or portion thereof, using a restriction site not present elsewhere in the construct and linking the 3xe2x80x2-most end of a nucleic acid fragment with the 5xe2x80x2 end of the vector, or portion thereof, using a restriction site not present elsewhere in the construct, and further linking the 3xe2x80x2 end of the 5xe2x80x2-most nucleic acid fragment with the 5xe2x80x2 end of the 3xe2x80x2-most fragment using a restriction site not present elsewhere in the construct, wherein each fragment specifies a component associated with at least one of a disease state, an environmental condition, or a biological organism wherein the component is different from a feature in any other component present elsewhere in the construct.
The invention includes a kit for producing a control DNA construct. The kit comprises a vector and at least two nucleic acid fragments, wherein the vector comprises at least two restriction sites that do not appear elsewhere in the construct, and wherein each the fragment comprises a restriction site at each end wherein the restriction site does not appear elsewhere in the construct but is complimentary with a restriction site at the end of another fragment or with an end of the vector, and wherein each fragment specifies a component of at least one of a disease state, an environmental condition, or a biological organism, wherein the feature is different from a component specified by any other fragment present elsewhere in the construct, the kit further comprising an applicator, and an instructional material for the use thereof.
In one aspect, the kit further comprises a restriction endonuclease specific for the restriction site.